Project Description Cholangiocarcinoma (CCA) is a highly malignant epithelial cancer of the biliary tree. The incidence and mortality of CCA is rising worldwide and currently there is no effective chemoprevention or treatment. The molecular pathogenesis underlying biliary epithelial neoplasia involves genetic and epigenetic changes leading to alterations of oncogenic and tumor suppressive pathways. While recent high throughput next-generation sequencing analyses have aided the identification of genetic abnormalities in CCA, the potential impact of epigenetic alterations on biliary epithelial neoplasia remains largely unknown. The current proposal is based on our exciting preliminary studies that EZH2 is a pivotal histone methyltransferase that epigenetically silences the expression of tumor suppressor miR-34a through H3K27 trimethylation in biliary cancer cells, and that miR-34a suppresses CCA cell growth by targeting Notch1, Notch2 and Jagged1. Our experimental findings support the hypothesis that the EZH2 histone methyltransferase promotes biliary carcinogenesis through epigenetic silencing of miR-34a and subsequent activation of Notch signaling, and that EZH2 inhibition or miR-34a replacement therapy in conjunction with standard chemotherapeutic regimen may represent an effective strategy for the treatment of human CCA. These hypotheses will be evaluated in two complementary Specific Aims. In Specific Aim 1, we will evaluate the effect and mechanism of the EZH2 histone methyltransferase in biliary carcinogenesis. Studies will be carried out to determine EZH2-regulated gene expression profile and its mechanism of action. The impact of EZH2 knockdown or overexpression on CCA development will be assessed in two complementary mouse models of cholangiocarcinogenesis (induced via transduction of the biliary tree and via hydrodynamic tail vein injection). The relevance of EZH2 and related signaling molecules will be validated in human CCA tissues and pre-cancerous bile duct lesions. In Specific Aim 2, we will evaluate the therapeutic efficacy of EZH2 inhibition or miR-34a replacement in conjunction with standard chemotherapy in pre-clinical models of CCA. The proposed studies will define the biological functions and molecular mechanisms of EZH2 and related signaling molecules in biliary carcinogenesis and lead to the development of new epigenetics-based target therapy.